2-feb-2023

1. Chaitanya K et al. International Research Journal Of Pharmacy, 2023, 14:02:1-5. Research Article INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230-8407 [LINKING] EXPERIMENTAL EVALUATION OF ANALGESIC EFFECT OF CLOVE OIL WITH COMPARISON OF TRAMADOL IN MICE Kandula Chaitanya,1 Sajja Suguna,2 Bhuvaneshwari Gajala,3 Edavaluru Shravya4 1Final year Postgraduate, Department of Pharmacology, Gandhi medical college, Secunderabad, Telangana 2Assistant Professor, Department of Pharmacology, Gandhi medical college, Secunderabad, Telangana 3Professor and HOD, Department of Pharmacology, Gandhi medical college, Secunderabad,Telangana 4Intern, Kamineni academy of medical sciences and research centre, LB Nagar, Telangana Address for correspondence Dr. Sajja Suguna E-mail: sugunasajja77@gmail.com Article Received: 05/01/2023, Article Accepted: 19/01/2023, Article Published: 16/02/2023 How To Cite: Chaitanya K, Suguna S,Gajala B,Shravya E. Experimental evaluation of analgesic effect of clove oil with comparison of tramadol in mice. International Research Journal of Pharmacy, 2023, 14:02:1-5. DOI: 10.56802/2230-8407.1303205 ABSTRACT: Background: Pain, the most common reason for patients, is an unpleasant sensation. It is generally caused by inflammatory reaction in the tissue or tissue damage and produced emotional reaction. Clove oil is an important aromatic spice, which belongs to the Myrtaceae family, contains high number of phenolic compounds with several biological activities, including analgesic, anticonvulsant, antibacterial, antifungal, insecticidal and antioxidant properties. Eugenol is the major compound in Clove essential oil. It is used as a local antiseptic and analgesic and anesthetic. Aim: To study the analgesic activity of clove oil in comparison to tramadol in mice. Methods: This experimental study was done in central animal house, Department of Pharmacology, Gandhi medical college, study duration from 18th august 2022 to 1st october . Three months old Male albino mice(n=15) were divided into three groups: 1) control group treated with normal saline, 2) standard group treated with inj.Tramadol 50mg/kg(Zydus corza zydus health are Ltd)., 3) Test group treated with Inj.clove essential oil 0.1 ml/kg (Dwibhashi bapamma Ayurvedic Nilayam Pvt.Ltd.) 15 min before testing in hot plate. Analgesic effect of clove oil and Tramadol had been seen with Hot plate method as a basic method to induce their respective doses. Repeated measures of ANOVA with Post-hoc Tukey’s HSD test were used for statistical analysis. Results: Repeated measures of ANOVA with Post hoc Tukey’s HSD test showed that maximum analgesic effect in group treated by 10% Clove essential oil was significantly higher (P value<0.05) than Normal saline group and similar to Tramadol group. Conclusion: Present study results showed that Clove oil may possibly act as analgesic agent from heat with stimulates the nociceptive mechanism. Clove essential oil has analgesic action similar to Tramadol in mice using hot plate test. Key words: Normal saline, Hot plate, Nociceptive, Phenolic compounds, Eugenol. INTRODUCTION Pain, the most common reason for patients, is an unpleasant sensation. It is generally caused by inflammatory reaction in the tissue or tissue damage and produces emotional reaction. At present most of the people determine to select medicinal plants to relief pain instead of drugs because it has fewer toxic effects, and can treat a variety of diseases1. Various herbal extracts can be used as analgesic remedies. Clove oil is an important aromatic spice, which belongs to the family Myrtaceae. Clove is cultivated in India, Madagascar, Sri Lanka and Malaysia2,3. Clove oil contains high number of phenolic compounds with several biological activities including 1

2. Chaitanya K et al. International Research Journal Of Pharmacy, 2023, 14:02:1-5. analgesic, anticonvulsant, antibacterial, antifungal, insecticidal and antioxidant properties, Eugenol is the major compound in CEO, with at least 50% of CEO. At least thirty compounds have been identified in the Clove Essential Oil (CEO)4,5,6,7.The remaining 10 to 40% of CEO are eugenyl acetate, β-caryophyllene, and α- humulene. Less than 10% corresponds to minor or trace components such as diethyl phthalate, caryophyllene oxide, cadinene, α-copaene, 4- (2-propenyl) -phenol, α-cubebene, γ-muurolen8,9. Eugenol (C10H12O2; 2- methoxy-4-(2-propenyl) phenol), is an allyl chain-substituted guaiacol. It is a clear, pale yellow, oily liquid which is slightly soluble in water and soluble in organic solvents. In dental plasters, fillings, and cements either clove oil or eugenol have been used for their topical analgesic properties10. In addition to these indications, it is used as a local antiseptic and anaesthetic. Current available literature states that 10%clove oil has potent analgesic activity in comparison to morphine, indomethacin, tramadol in combination, but not in comparison to tramadol alone as it doesn’t produce dependency in comparison to above mentioned analgesic drugs. Therefore, the Present study had been planned to evaluate the Analgesic activity of 10% clove oil in mice in comparison with Tramadol. MATERIALS AND METHODS The present study deign is an Experimental and comparative, and this was conducted in Central animal house, Department of Pharmacology, Gandhi medical college, study duration from 18th august 2022 to 1st October 2022. [After the approval of IAEC and IAEC Reg. no. 28/GMC/IAEC/428/GO/Re/S/2001/CPCSEA] ANIMALS AND DRUGS: Male albino mice (n=15), three months old, weighing 30 grams that were used. All mice were housed in 4-6 mice per standard cage room temperature (23±1°C) on a 12 h light/dark cycle. Food was withheld overnight before the experiments while water was still provided. All experiments were done during the day time. The experimental protocol dated 18th February 2022, was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) under Ref.No.28/GMC/IAEC/428/GO/Re/S/2001/CPCSEA, as per CPCSEA guidelines animal handling and further procedures to evaluate analgesic effect of clove oil in mice have done in accordance with Gandhi Medical College, Animal Ethical Committee Acts, Reg, No:428/GO/Re/S/2001/CPCSEA. The clove oil was bought from Dwibhashi Bapamma Ayurvedic Nilayam Pvt. Ltd. The composition of the clove oil was 100% natural, and Tramadol was from (Zydus corza zydus health care Ltd). No other chemicals or anaesthetic agents used to evaluate analgesic activity of test drugs. Study medication had been tested in albino mice those already in facility with 3 days of acclimatization period for physiologic, psychologic and nutritional stabilization prior to experimental evaluation. EXPERIMENTAL DESIGN:11 Pilot study was performed in total number of animals (n=6) mice in each group n=2 mice was used to analyze analgesic action of clove oil compared to tramadol for basic Evaluation of analgesic effect of clove oil had been seen in n=15 mice which was divided into three groups: Control group(n=5), Standard group(n=5), test group(n=5). Group 1 treated with Normal saline 5 ml/kg, Group2 Standard treated with Inj. Tramadol 50 mg/kg, Group3-Test Treated with Inj. Clove oil 0.1ml/kg 15 mins before testing in hot plate. The volume of Injection induced 10 ml/kg (i.p.). NOCICEPTIVE TEST: 11 To assess nociceptive response hot plate method was used as it is a feasible and standardized method to test effectiveness I of analgesic in basic pain research. In hot platemethod, animals were placed on the hot plate with temperature setting controlled at 55±0.2 ° C and the Cut-off time was 60 seconds. Nociceptive response was defined aslicking forepaws. Time duration between placing the animals on hot plate and licking forepaws were considered as reaction time. The hot plate was performed in seven(n=7) readings with 15 min interval from base line to 90 mins. All three groups were subjected to above mentioned time duration with the respective dosage of drugs. STATISTICAL ANALYSIS: Analgesic effect of Normal saline, Tramadol and Clove oil were observed. The obtained data presented as Mean ±SD. Statistical comparison of Basal reaction time between three groups was done with One way analysis of Variance ANOVA with Post-hoc Tukey’s HSD test in Microsoft excel. Difference was considered statistically significant when p=<0.05. RESULTS In the present day, it was demonstrated Analgesic effect of clove oil with comparison to tramadol in Mice using Hot plate method. The study animals were grouped into three and they were subjected to respective doses of study medication and then placed on the hot plate with temperature setting Controlled at 55 ±0.2 C. Cut-off 2

3. Chaitanya K et al. International Research Journal Of Pharmacy, 2023, 14:02:1-5. time was 60 seconds. The basal reaction time in mean±SD between three study groups from base line time 0 to 90 mins had been shown in Table no 1. As it is mentioned below table no1/ figure no1- Comparison between groups shows maximum analgesic effect with 10% Concentration of clove oil (test group) was higher than tramadol (standard group) and normal Saline (Control group). In Present study observed basal reaction time between the different study groups from Base line to 90 mins duration with Mean±SD. Control group showed minimum response at Base line time with 13±0.63 and maximum response at 90 mins with 15.6±0.49. Standard group showed minimum response at Base line time with 13.2±0.74 and Maximum response at 90 mins with 18.2±0.74. Test group showed minimum response at Base line time with 13.6±1.02 and Maximum response observed at 90 mins with 19.4±1.02. The maximum effect of study medication in three different study groups had been compared applying ANOVA with Post-hoc Tukey’s HSD test i.e., shown in Table no.2,3,4 and figure no.1 Repeated measures of ANOVA with Post-hoc Tukey’s HSD test showed that maximum analgesic effect in three groups’ animals treated by Normal saline, Inj. Tramadol 50 ml/kg, Inj. Clove oil (10%) 0.1 ml/kg, shown in figure no.1 Among these animals treated by Clove oil (Test group) was significantly higher than Normal saline (Control group) and similar to Tramadol (Standard group) when P value < 0.05, shown in table no.4 DISCUSSION In the present study Analgesic effect of clove oil was evaluated using Hot plate method in comparison to Tramadol by measuring heat threshold in mice. In hot plate method, animals were placed on the hot plate with temperature setting controlled at 55 ±0.2C. The unrestrained animals were placed on the plate until nocifensive behaviour is observed and the cut-off time was 60 seconds. The nocifensive response was observed i.e., Licking forepaws or withdrawal and moving hind paws, during study procedure if the animals were not reactive to test, they had been removed after above mentioned time point to avoid tissue damage in animal. The present study results states that 10% concentration of clove oil had been showed maximum effectiveness which was similar to study reported by Hossieni et al. conducted a study on analgesic effect of clove oil in Mice using hot plate Method, showed that 10% concentration of clove oil was the most effective in Mice comparison with other two doses of clove oil (2%,5%) concentrations 11. Kamakar Asl et al. done a study on Analgesic effect of the aqueous and ethanolic extract of clove oil in mice, observed maximum nociceptive effect in animal groups treated with 50mg/kg of Clove oil was significantly higher in mice than the Naloxone dose 100mg/kg, 200 mg/kg treated animals12, which is similar to present study results i.e.,10% clove oil is more analgesic effect than tramadol 50mg/kg. A study done by Pouya Tayebi et al. Tramadol effect on morphine dependency and analgesia in mice, concluded analgesic effect of Tramadol doses of 25 mg/kg; 50 mg/kg was higher in mice than Morphine 10 mg/kg13. In the present study Comparison between groups shows maximum analgesic effect with 10% Concentration of clove oil (test group) was little more during the experiment with tramadol (standard group) and normal Saline (Control group) but statistically it shows insignificant to prove statistical significance may require more sample size. Kanyarat sueksakit et al. conducted a study of syzygium aromaticum on analgesic activity in rats, observed analgesic effect of Indomethacin 5% w/w was higher with tail flick method in rats than clove oil of different doses (2%,5%,10%)14. A study conducted by Yousef A. Taher et al. experimental evaluation of anti- inflammatory, antinociceptive and antipyretic activities of clove oil in mice, concluded nociceptive effect of morphine 5mg/kg was significantly higher in mice than clove oil 0.03% 15. The analgesic effect of eugenol has been attributed to its capability for inhibiting the prostaglandins and other inflammatory mediators such as leukotrienes 16. The analgesic studies revealed that clove oil exhibited potent analgesic effect against tramadol12, clove oil may possibly act as analgesic agent from heat with stimulates the nociceptive mechanism. Hence, the analgesic effect of clove essential oil which was shown in the present study should be considered for further evaluation. CLINICAL IMPLICATIONS: Clove oil may have benefits for dental and topical application for treating headache, ear pain, tooth ache, infections and even fighting cancer pain. 3

4. Chaitanya K et al. International Research Journal Of Pharmacy, 2023, 14:02:1-5. LIMITATIONS  Sample size is the one of the limitation, Hence the present study had been planned for basic evaluation of analgesic effect of 10% clove oil.  Analgesic effect to be evaluated with multiple doses of clove oil for further clinical implication CONCLUSION Present study results showed that analgesic effect of 10% concentration of clove essential oil is effective like Tramadol in mice. The role of clove oil and its analgesic effect which was shown in the present study should not be ignored. However, clove is an aromatic spice and high number of phenolic compounds with several biological activities needed to be investigated further to know its exact mechanism for clinical implications. REFERENCES: 1. Kanyarat Sueksakit, preliminary study of syzygium aromaticum on analgesic activity in rats, Thai J. Pharm. Sci. 2013;38:63-65. 2. Arung ET, Matsubara E, Kusuma IW, Sukaton E, Shimizu K, Kondo R. Inhibitory components from the buds of clove (Syzygium aromaticum) on melanin formation in B16 melanoma cells. Fitoterapia. 2011;82(2):198- 202. 3. Tyler VE, Brady LR, Robbers JE. Pharmacognosy. Philadelphia, PA, USA: Lea and Febriger; 2016;7(1):1-6. 4. Tunç, M.T.; Koca, İ. Ohmic heating assisted hydrodistillation of clove essential oil. Ind. Crops Prod. 2019; 14:111763. 5. Guan, W.; Li, S.; Yan, R.; Tang, S.; Quan, C. Comparison of essential oils of clove buds extracted with supercritical carbon dioxide and other three traditional extraction methods. Food Chem. 2007; 10:1558–1564. 6. Golmakani, M.-T.; Zare, M.; Razzaghi, S. Eugenol Enrichment of Clove Bud Essential Oil Using Different Microwave-assisted Distillation Methods. Food Sci. Technol. Res. 2017; 23:385–394. 7. Hatami, T.; Johner, J.C.F.; Zabot, G.L.; Meireles, M.A.A. Supercritical fluid extraction assisted by cold pressing from clove Preprints, not peer-reviewed:Extraction performance, volatile oil composition, and economic evaluation. J. Supercrit. Fluids 2019; 144:39–47. 8. Bakkali, F.; Averbeck, S.; Averbeck, D.; Idaomar, M. Biological effects of essential oils – A review. Food Chem. Toxicol. 2008; 46:446–475. 9. Bakry, A.M.; Abbas, S.; Ali, B.; Majeed, H.; Abouelwafa, M.Y.; Mousa, A.; Liang, L. Microencapsulation of Oils: A Comprehensive Review of Benefits, Techniques, and Applications. Compr. Rev. Food Sci. Food Saf. 2016; 15:143–182. 10. Chaieb K, Hajlaoui H, Zmantar T, Kahla-Nakbi AB, Rouabhia M, Mahdouani K, Bakhrouf .The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytotherapy Research, 2021; 501-506. 11. Mahmoud Hosseini, Mina Kamkar Asl, Hassan Rakhshandeh, Analgesic effect of clove essential oil in mice. Avicenna Journal of Phytomedicine.2011;1(1):1-6 12. Mina Kamkar Asl, Ashraf Nazariborun, Mahmoud Hosseini, Analgesic effect of the aqueous and ethanolic extracts of clove, Avicenna Journal of Phytomedicine.2013;2(3):186-192. 13. Pouya Tayebi, Farzan Kheirkhah, Gouya Tayebi, Tramadol Effect on Morphine Dependency and Analgesia in Mice, International Journal of Pharmacology.2008;4(6):452-459. 14. Kanyarat Sueksakit, Krittiya Thisayakorn, Vichein Khueynok , Kanjana Sriyam, Darunee Pahusee , and Nopparat Buddhakala, preliminary study of syzygium aromaticum on analgesic activity in rats, thai j. Pharm. Sci. 2013;38:63-65. 15. Yousef A Taher, Awatef M Samud,Fathy E El-Taher, Ghazala ben-Hussin, Badryia F Al- Mehdawi, Hanan A Salem, Experimental evaluation of anti-inflammatory, antinociceptive and antipyretic activities of clove oil in mice.2015;10:28685. 16. Raghavenra H, Diwakr BT, Lokesh BR, Naidu KA.2006. Eugenol--The active principle from cloves inhibits 5-lipoxygenase activity and leukotriene-C4 in human PMNL cells. Prostaglandin Leukot Essent Fatty Acids,74: 23-27. TABLES 4

5. Chaitanya K et al. International Research Journal Of Pharmacy, 2023, 14:02:1-5. Study groups Baseline 15 mins Control group 13±0.632 13.4±0.49 Standard group 13.2±0.74 14.2±0.74 Test group 13.6±1.02 15.4±1.02 30 mins 13.6±0.49 14.6±0.74 16.2±1.32 45 mins 14.6±0.49 15.8±0.74 17.2±1.16 60 mins 14.2±1.16 16.6±0.8 18.4±1.02 75 mins 15.4±0.8 17.8±0.74 18.2±0.74 19±0.632 90 mins 15.6±0.49 19.4±1.02 Table 1; Basal reaction time between the different study groups from Base line to 90 mins duration with Mean±SD. Groups Mean±SD Standard error Normality Control group 14.2±0.99 Standard group 15.8±1.85 Test group 17±2.08 Table 2; Comparison of analgesic effect of study medication applying ANOVA with Post-hoc Tukey’s HSD test. Table Source DF Sum of Square Mean Square F Statistic P-Value Groups (Between groups) 2 26.4686 Error (Within groups) 18 52.6971 Total 20 79.1657 0.37 0.70 0.78 0.7065 0.9171 0.8896 3; 13.2343 2.9276 3.9583 4.5205 0.02566 Repeated measures of ANOVA with Post-hoc Tukey’s HSD test showed that maximum analgesic effect showed with significant P-value (0.02566). Treatments pair Control Vs Standard group 2.3857 Control Vs Test group Standard Vs Test group Tukey HSD Q statistic Tukey HSD p-value Tukey HSD inference 0.2372168 4.2413 0.0199883 1.8556 0.4082317 Insignificant P<0.05 Significant Insignificant Table 4; Post hoc Tukey’s HSD test applied within the study groups (P<0.05) Figure 1; Comparison of analgesic effect of study medication in three study groups 5